Proteins that are present on the surface of a cell or virus are typically transmembrane proteins. These proteins include cell surface receptors and envelope glycoproteins, and these proteins are involved in a variety of-protein to protein interactions. For example, as demonstrated with pseudotyped viral particles, the specific envelope protein present on a viral surface determines the receptor that the virus will bind to. Additionally, the three dimensional conformation of the protein has an important effect on the particular interaction. However, maintaining a desired conformation can be difficult. For example, many receptors, envelope proteins, etc. are the result of multimeric formation of individual monomers. Thus, even though each subunit may span the membrane only once, the multimeric complex has several membrane-spanning components that could contribute to its overall conformational integrity.
Significant attention has been focused on viruses including the flu virus, herpes virus, retroviruses, lentiviruses, etc., particularly in the mechanism of infection. Human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2) are the etiologic agents of acquired immunodeficiency syndrome (AIDS), which results from the profound depletion of CD4-positive lymphocytes in infected individuals (Barre-Sinoussi, F., et al., Science 220:868-71, 1983; Gallo, R. C., et al., Science 224: 500-3, 1984; Fauci, A. S., et al., Ann Intern Med 100: 92-106, 1984).
Though great progress has been made in the treatment of individuals infected with viruses such as HIV, numerous problems still remain. For example, treatment typically requires taking cocktails of medicines at different times over extended periods of time. The failure to do so can result in seriously undermining the treatment, and ultimately results in further progression of the disease. Even where individuals follow the treatment protocol, there are many instances of disease progression. Moreover, the treatment is extremely costly, effectively rendering it out of reach to many individuals in the United States, and in much of the rest of the world. There are also other viruses for which antiviral therapy has not yet been developed.
Accordingly, the development of alternative methods of dealing with viral infection, such as HIV infection, is still extremely important.
One area where a great deal of attention has been extended has been in utilizing viral sub-units to generate immune reactions. Antibodies that neutralize viruses typically do so by inhibiting viral binding to surface receptors. The major protein found on the surface of HIV, and therefore a major target for generating neutralizing antibodies, is the envelope glycoprotein, gp120. This protein appears on the surface of the virion, thus rendering it a prime target for the immune system.
Unfortunately, the HIV-1 envelope glycoproteins have proven inefficient in generating antibodies that neutralize the virus, especially those that can neutralize more than a limited number of HIV-1 strains (Berman, P. W., et al., J. Infect. Dis. 176: 38497, 1997; Connor, R. I., et al., J. Virol. 72: 1552-76, 1998; Mascola, J. R., et al., J. Infect. Dis. 173: 340-8, 1996; reviewed in Burton, D. R. and D. C. Montefiori, AIDS 11 Suppl. A: S87-98, 1997; Burton, D. R. and J. P. Moore, Nature Med. 4(5 Suppl.) 495-8, 1998; and Wyatt, R. and J. Sodroski, Science 280: 1884-8, 1998). Many of the antibodies elicited by the envelope glycoproteins are not able to bind efficiently to the functional envelope glycoprotein trimer and therefore are devoid of neutralizing activity (Broder, C. C., et al., Proc. Natl. Acad. Sci. USA 91: 11699-703, 1994; Moore, J. P., et al., J. Virol. 69: 101-9, 1995; Moore, J. P., et al., J. Virol. 70: 1863-72, 1996; Parren, P. W., et al., Nature Med. 3:366-7. 1997; Parren, P. W., et al., J. Virol. 72: 3512-9, 1998; Wyatt, R., et al., J. Virol. 71: 9722-31, 1997). The lability of the envelope glycoprotein trimers, conformational flexibility in the shed gp120 glycoprotein, and the variability and glycosylation of the gp120. surface all appear to contribute to the poor neutralizing antibody responses (reviewed in Montefiori, D. C., et al., AIDS Res. Human Retroviruses 15: 689-98, 1999; Moore, J., et al., J. Virol. 68: 469-84, 1995; and Wyatt, R., and J. Sodroski, Science 280: 1884-8, 1998).
The entry of primate lentiviruses such as HIV-1 and HIV-2 into target cells is mediated by the viral envelope glycoproteins (Wyatt, R., and J. Sodroski, Science 280: 1884-8, 1998). The mature envelope glycoproteins on the primate lentivirus are organized into an external gp120 (gp125 for HIV-2) exterior envelope glycoprotein and the gp41 transmembrane envelope glycoprotein (gp36 for HIV-2) (Alan, J. S., et al., Science 228: 10914, 1985; Earl, P. L., et al., J. Virol. 65: 2047-55, 1991; Robey, W. G., et al., Science 228: 593-595, 1985; Veronese, F. D., et al., Science 229: 1402-1405, 1985; Wyatt, R., and J. Sodroski, Science 280: 1884-8, 1998). For example, in the infected cell, the HIV-1 envelope glycoprotein is initially synthesized as an 845- to 870-amino acid protein, depending upon the viral strain (Earl, P. L., et al, J. Virol. 65: 2047-2055, 1991). N-linked, high-mannose sugars are added to this primary translation product to result in the gp160 envelope glycoprotein precursor (gp140 for HIV-2). Oligomers of gp160 form in the endoplasmic reticulum, and several pieces of evidence suggest that these are trimers. First, X-ray crystallographic studies of fragments of the gp41 ectodomain revealed the presence of very stable, six-helix bundles (Chan, D. C., et al., Cell 89: 263-73, 1997; Tan, K., et al, Proc. Natl. Acad. Sci. USA 94: 12303-8,1997; Weissenhorn, W., et al., Nature 387: 426-30,1997). These structures were composed of a trimeric coiled coil involving N-terminal gp41 α helices, with three C-terminal gp41 α helices packed into the grooves formed by the three inner helices. Second, introduction of cysteine pairs at specific locations in the coiled coil resulted in the formation of intermolecular disulfide bonds between the gp160 subunits (Farzan, M., et al, J. Virol. 72: 7620-5, 1998). The disulfide-stabilized oligomer was shown to be a trimer. Finally, the matrix proteins of HIV-1 and the related simian immunodeficiency viruses, which interact with the intravirion domains of the envelope glycoproteins, crystallize as trimers (Hill, C. P., et al., Proc. Natl. Acad. Sci. USA 93: 3099-3104, 1996; Rao, Z., et al., Nature 378: 743-7, 1995).
Following oligomerization, the precursor glycoprotein is transported to the Golgi apparatus, where cleavage by a cellular protease generates the external protein, gp120, and the trans-membrane protein, gp41 (Alan, J. S., et al., Science 228: 1091-4, 1985; Robey, W. G., et al., Science 228: 593-595, 1985; Veronese, F. D., et a., Science 229: 1402-1405, 1985). The gp120 glycoprotein remains associated with the gp41 glycoprotein through non-covalent, hydrophobic interactions (Helseth, E., et al., J. Virol. 65:2119-23, 1991; Kowalsid, M., et al, Science 237: 1351-1355, 1987). The lability of the gp120-gp41 association results in the “shedding” of some gp120 molecules from the trimer, resulting in non-functional envelope glycoproteins (McKeating, J. A., et al., J. Virol. 65: 852-60, 1991; Willey et al., J. Virol. 68: 1029-39, 1994). It has been suggested that these disassembled envelope glycoproteins result in the generation of high titers of non-neutralizing antibodies during natural HIV-1 infection (Burton, D. R., and J. P. Moore, Nat. Med. 4 (5 Suppl.): 495-8, 1998; Moore, J. P., and J. Sodroski, J. Virol. 70 1863-72, 1996; Parren, P. W., et al., J. Virol. 72: 3512-9). The envelope glycoprotein trimers that remain intact undergo modification of a subset of the carbohydrate moieties to complex forms before transport to the cell surface (Earl, P. L., et al., J. Virol. 65: 2047-55, 1991).
The mature envelope glycoprotein complex is incorporated from the cell surface into virions, where it mediates virus entry into the host cell. The gp120 exterior envelope glycoprotein binds the CD4 glycoprotein, which serves as a receptor for the virus (Dalgleish, A. G., et al., Nature 312: 763-7, 1984; Klatzmann, D., et al., Nature 312: 767-8, 1984; McDougal, J. S., et al, J. Immunol. 137: 2937-44, 1986). Binding to CD4 induces conformational changes in the envelope glycoproteins that allow gp120 to interact with one of the chemokine receptors, typically CCR5 or CXCR4 (Alkhatib, G., et al., Science 272: 1955-8, 1996; Choe, H., et al., Cell 85: 113548, 1996; Deng, H., et al., Nature 381: 661-6, 1996; Doranz, B. J., et al., Cell 85: 1149-58, 1996; Dragic, T., et al., Nature 381: 667-73, 1996; Feng, Y., et al., Science 272: 872-7, 1996; reviewed in Choe, H., et al., Semin. Immunol. 10: 249-57, 1998). The chemokine receptors are seven-transmembrane, G protein-coupled receptors, and gp120 interaction with the chemokine receptors is believed to bring the viral envelope glycoprotein complex nearer to the target cell membrane and to trigger additional conformational changes in the envelope glycoproteins. Although the exact nature of these changes is unknown, mutagenic data are consistent with a role for the hydrophobic gp41 amino terminus (the “fusion peptide”) in mediating membrane fusion (Cao, J., et al., J. Virol. 67: 2747-55, 1993; Freed, E. O., et al., Proc. Natl. Acad. Sci. USA 87: 4650-4, 1990; Helseth, E., et al., J. Virol. 64: 6314-8, 1990; Kowalsid, M., et al., Science 237: 1351-5, 1987). It has been suggested that, following interaction of the “fusion peptide” with the target cell membrane, formation of the six-helical bundle by the three gp41 ectodomains would result in the spatial juxtaposition of the viral and target cell membranes (Chan, D. C., et al., Cell 89: 263-73, 1997). Six-helical bundles have been documented in several viral envelope glycoproteins that mediate membrane fusion and virus entry (Bullough, P. A., et al., Nature 371: 37-43, 1994; Carr, C. M., and P. S. Kim, Cell 73: 823-32, 1993; Weissenhorn, W., et al., Proc. Natl. Acad. Sci. USA 95: 6032-6, 1998; Weissenhorn, W., et al., Mol. Cell 2: 605-16, 1998). The formation of this energetically stable structure from a different and as-yet-unknown precursor structure is believed to provide the energy necessary to overcome the repulsion between the viral and cell membranes.
Initial attempts to generate immune reactions to HIV envelope glycoproteins have encountered substantial difficulties. For example, it was discovered that there are numerous regions in the glycoprotein which rapidly mutate in response to antibodies or drugs directed thereto. These regions also vary significantly from one strain of HIV to another. Accordingly, these regions have been described as variable regions. There are other regions that are conserved among HIV-1, HIV-2 and SIV strains. Variable regions and conserved regions of gp120 have been mapped and are well known in the art. In the three-dimensional structure of the protein, these variable regions are typically at the surface, and thus mask the more conserved regions. The variable regions are highly antigenic, typically generating most of the antibodies seen. It is only late in the progression of the disease that antibodies generated to the conserved regions are typically seen. Such antibodies include the F105 antibody, the 17b antibody and the 48d antibodies. The amino acids comprising the epitopes for these antibodies are proximal to each other in the three-dimensional structure of the protein, but appear distant from each other when one looks strictly at a one-dimensional linear amino acid sequence. Such an epitope is referred to as a discontinuous conformational epitope. Furthermore, the amino acids comprising these discontinuous conformational epitopes are located in a number of conserved regions. Numerous variable-region deleted glycoproteins that expose these discontinuous conformational epitopes by deleting portions of the variable regions are disclosed in U.S. Pat. Nos. 5,817,316 and 5,858,366.
Consequently, it is clear that the three-dimensional structure of the protein is extremely important in terms of what the immune system actually sees. Unfortunately, the individual monomers like other multimeric proteins do not typically form a stable multimer, in this case trimeric spikes, that approximate the natural wild type confirmation. Thus, generating neutralizing antibodies depends upon stabilizing the three-dimensional, trimeric structure of the envelope glycoprotein.
Attempts have been made to stabilize trimers by stabilizing interactions in the gp41 segment, for example by introducing cysteine residues. Another approach has been by inserting coiled coils in a portion of the transmembrane protein such as for HIV-1, gp41 or for HIV-2, gp36. Given the importance of being able to make and use such stable multimers, it is very desirable to have new methods for preparing such stable trimers. Particularly so if one wants to use such multimers to elicit an immune response.
The ability to create new in vitro assays to screen for molecules that can interact with a stable multimer such as the envelope glycoprotein is extremely important.
The G protein-coupled seven transmembrane segment receptor CXCR4, previously called HUMSTR, LCR-1 or LESTR (Federsppiel et al., 1993; Jazin et al., 1993; Loetscher et al., 1994) has been shown to allow a range of non-human, CD4-expressing cells to support infection and cell fusion mediated by laboratory-adapted HIV-1 envelope glycoproteins (Feng et al., 1996). Other G-protein-coupled seven transmembrane segment receptors such as CCR5, CCR3 and CCR2 have been shown to assist cellular entry of other HIV-1 isolates. It is believed that the cellular entry occurs as a result of the interaction of the external envelope glycoprotein, e.g., gp120, CD4 and the chemokine receptor. This further illustrates the importance of having an in vitro screen for testing molecules that more closely approximates the wild type env to determine their effect on the external env. Thus, the ability to express the envelope glycoprotein with its three-dimensional structure is extremely important in terms of what the immune system actually sees.
One of the particular challenges in expressing the env protein with its wild type conformation is that it is a transmembrane protein. Transmembrane proteins or integral membrane proteins are amphipathic, having hydrophobic domains that pass through the membrane and interact with the hydrophobic lipid molecules in the interior of the bilayer, and hydrophilic domains which are exposed to the aqueous environment on both sides of the membrane (for example, the aqueous environments inside and outside of the cell). The biological activities of integral membrane proteins (e.g., ligand binding) can be dependent upon the hydrophilic domains; in some cases, the membrane—spanning regions contribute to function.
It would be desirable to produce, isolate and stabilize in purified form while retaining their wild-type conformation transmembrane proteins, particularly multimeric proteins, such as gp120, and oligomeric complexes of transmembrane proteins such as gp120/CD4 and gp120/CD4/CCR5. It would be desirable if these proteins could be maintained in their wild-type conformation for extended periods of time and under conditions commonly found in vivo. The purification of transmembrane proteins, including oligomeric complexes, in a functionally relevant conformation should expedite the ability to elicit immune reactions to epitopes specifically exposed when the protein(s) is in its the wild-type conformation or in an oligomeric complex, as well as expedite their use in screening assays to identify antibodies, ligands, and small molecules that bind the transmembrane protein(s).